The project is aimed at initiating a research collaboration between the laboratories of the PI and foreign investigator. The PI's parent project places emphasis on the poly-phosphoinositide (poly-PI) signaling pathway in neuronal damage after chronic alcohol consumption. The collaborator's laboratory is active in studying cerebral injury mediated by nitric oxide synthase (NOS). The goal for this project is to test the hypothesis that chronic ethanol consumption alters the muscarinic cholinergic receptor-mediated poly-PI signaling activity and NOS activity in the brain. The project will use the brain slice preparation. The first aim is to examine the effects of neural NOS activity, its protein and mRNA in different brain regions of C57BL/6J mice and relate these changes to IP3 level, IP3R binding, and IP3R mRNA in the same brain regions. In addition, changes in nNOS activity will be examined at different times after ethanol withdrawal. These studies are designed to provide basic information, lacking at present, about the effect of chronic ethanol on NOS activity in the brain. Mice will be given an ethanol diet for a period of weeks and tested for the development of tolerance using sleep-time and righting reflex after ip injection of ethanol (this in collaboration with Professor W. Kostowski in Warsaw). Mice will be euthanized and brain tissue (including areas where cholinergic pathways are well represented) will be evaluated for determination of nNOS activity, protein (Western blot) and mRNA (in situ hybridization); IP3 levels, binding, proteins and mRNA. These parameters will be evaluated in ethanol tolerant mice and at 4, 8, and 24 hours after withdrawal. The combined laboratories have the expertise and resources for all procedures. The second aim is to test the hypothesis that alteration of nNOS activity upon chronic ethanol administration is linked to the muscarinic cholinergic receptor-mediated poly-PI signaling pathway. Previous studies by the investigators have shown that chronic ethanol affects poly-PI signaling in mouse cerebral cortex and hippocampus, suggesting that nNOS activity in cholinergic muscarinic neurons (well represented in cortex) may be affected. The ability of cholinergic agonists to stimulate NOS activity will be studied and these findings extended to the study of brain slices in mice exposed to chronic ethanol at different times after withdrawal.